RESUMEN
Biomolecular reagents that enable the specific molecular recognition of proteins play a crucial role in basic research as well as medicine. Up to now, antibodies (immunoglobulins) have been widely used for this purpose. Their predominant feature is the vast repertoire of antigen-binding sites that arise from a set of 6 hypervariable loops. However, antibodies suffer from practical disadvantages because of their complicated architecture, large size, and multiple functions. The lipocalins, on the other hand, have evolved as a protein family that primarily serves for the binding of small molecules. Here, we show that an engineered lipocalin, derived from human Lcn2, can specifically bind the T cell coreceptor CTLA-4 as a prescribed protein target with subnanomolar affinity. Crystallographic analysis reveals that its reshaped cup-like binding site, which is formed by 4 variable loops, provides perfect structural complementarity with this "antigen." Furthermore, comparison with the crystal structure of the uncomplexed engineered lipocalin indicates a pronounced induced-fit mechanism, a phenomenon so far considered typical for antibodies. By recognizing the same epitope on CTLA-4 that interacts with the counterreceptors B7.1/B7.2 on antigen-presenting cells the engineered Lcn2 exhibits strong, cross-species antagonistic activity, as evidenced by biological effects comparable with a CTLA-4-specific antibody. With its proven stimulatory activity on T cells in vivo, the CTLA-4 blocking lipocalin offers potential for immunotherapy of cancer and infectious disease. Beyond that, lipocalins with engineered antigen-binding sites, so-called Anticalins, provide a class of small ( approximately 180 residues), structurally simple, and robust binding proteins with applications in the life sciences in general.
Asunto(s)
Antígenos CD/metabolismo , Epítopos , Lipocalinas/metabolismo , Ingeniería de Proteínas , Proteínas de Fase Aguda/genética , Anticuerpos/química , Antígenos CD/química , Sitios de Unión , Antígeno CTLA-4 , Cristalografía por Rayos X , Humanos , Indicadores y Reactivos/síntesis química , Indicadores y Reactivos/química , Lipocalina 2 , Lipocalinas/química , Lipocalinas/genética , Unión Proteica , Conformación Proteica , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Fas ligand (FasL) is a potent proapoptotic type-II transmembrane protein that can cause cell death in Fas+ target populations. Despite the presumed "silent" nature of apoptotic cell death, forced expression of FasL can induce a dramatic inflammatory response. To elucidate the in vivo mechanism(s) linking FasL and inflammation, we used a membrane-bound cell-free form of FasL (mFasL-vesicle preparation (VP)). We found that i.p. injection of FasL-microvesicles led to the rapid activation and subsequent demise of Mac1(high) resident peritoneal macrophages. Apoptosis of Mac1(high) peritoneal macrophages was observed within 0.5 h of mFasL-VP injection and correlated with the detection of increased macrophage inflammatory protein (MIP)-2 levels in peritoneal lavage fluid as well as induced RNA expression of IL-1beta, MIP-2, MIP-1alpha, and MIP-1beta. In vitro culture of purified peritoneal populations identified Mac1(high) cells as the major cytokine/chemokine producers in response to mFasL-VP. Purified Mac1(high) cells exposed to FasL could restore the ability of Fas-deficient mice to mount an inflammatory response. Our data demonstrate that the FasL-mediated inflammatory response starts with the production of proinflammatory mediators by preapoptotic resident tissue macrophages and suggest a general mechanism responsible for neutrophil inflammation seen in cases of FasL-expressing allografts.
Asunto(s)
Apoptosis/inmunología , Factores Quimiotácticos/biosíntesis , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Glicoproteínas de Membrana/metabolismo , Neutrófilos/inmunología , Neutrófilos/metabolismo , Receptor fas/metabolismo , Animales , Apoptosis/genética , Líquido Ascítico/inmunología , Líquido Ascítico/metabolismo , Líquido Ascítico/patología , Separación Celular , Quimiocinas/biosíntesis , Quimiocinas/genética , Quimiocinas/metabolismo , Factores Quimiotácticos/genética , Citocinas/biosíntesis , Citocinas/genética , Citocinas/metabolismo , Vesículas Citoplasmáticas/inmunología , Proteína Ligando Fas , Femenino , Regulación de la Expresión Génica/inmunología , Inflamación/inmunología , Leucemia L5178 , Ligandos , Macrófagos Peritoneales/patología , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos A , Ratones Endogámicos C3H , Ratones Endogámicos DBA , Ratones Endogámicos MRL lpr , Ratones Mutantes , Trasplante de Neoplasias , Proteínas Nucleares , Factores de Transcripción , Receptor fas/genéticaRESUMEN
Bioactive Fas ligand (FasL)-expressing vesicles were generated (vesicle preparation, VP) from two cell lines overexpressing FasL. The effect of NOK-1 anti-FasL mAb (mouse IgG1) on the cytotoxicity of FasL VP against various targets was determined. At high concentrations (1-10 microg/ml), NOK-1 inhibited the cytotoxicity. By contrast, NOK-1 in the dose range of 1-100 ng/ml significantly enhanced cytotoxicity against the FcR(+) LB27.4, M59, and LF(+) targets, but not the FcR(-) Jurkat and K31H28 hybridoma T cell targets. The ability to enhance FasL VP-mediated cytotoxicity could be blocked by the FcR-specific mAb 2.4G2. Enhancement was also observed with FcR(+) A20 B lymphoma but not with the FcR(-) A20 variant. Enhancement of FasL VP cytotoxicity was observed with five IgG anti-FasL mAbs, but not with an IgM anti-FasL mAb. Inhibition was observed with high doses of all mAb except the IgG anti-FasL mAb G247-4, which is specific to a segment outside the FasL binding site. Interestingly, under identical conditions but in the presence of 2.4G2, G247-4 inhibited the cytotoxicity of FasL VP. In addition, G247-4 inhibited the FasL VP-mediated killing of FcR(-) Jurkat. The data demonstrate that FasL-expressing bioactive vesicles display a property heretofore unknown in bioactive agents that express FasL-mediated cytotoxicity. The mechanism of the Ab-mediated, FcR-dependent enhancement of cytotoxicity of bioactive vesicles and its physiological significance are discussed.
Asunto(s)
Adyuvantes Inmunológicos/fisiología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Sistema Libre de Células/inmunología , Glicoproteínas de Membrana/biosíntesis , Receptores Fc/fisiología , Receptor fas/metabolismo , Animales , Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Especificidad de Anticuerpos , Línea Celular , Sistema Libre de Células/metabolismo , Pruebas Inmunológicas de Citotoxicidad , Proteína Ligando Fas , Humanos , Inmunoglobulina G/genética , Isotipos de Inmunoglobulinas/fisiología , Inmunosupresores/farmacología , Leucemia L5178 , Ligandos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Ratones , Receptores Fc/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacología , Células Tumorales CultivadasRESUMEN
Fas ligand (FasL) has been shown to mediate both apoptotic and inflammatory reactions. To rigorously assess the physiological role of different forms of the FasL molecule with regard to these two distinct processes, we isolated stably transfected lymphoma cell lines that expressed either murine wild-type FasL, membrane-only FasL, or functionally distinct forms of soluble FasL. First, the ability of these lines to induce an inflammatory response was assessed in vivo by injecting the transfectants intraperitoneally and measuring subsequent neutrophil extravasation into the peritoneal cavity. Second, lines were assessed by injecting the transfectants subcutaneously and monitoring their growth as solid tumors. Our study clearly demonstrated that the extent of inflammation induced by the transfectants directly correlated with their relative cytotoxic activities. A neutrophil response could only be elicited in mice with intact Fas death domains although Fas expression by the neutrophils was not essential. Lymphoma cells expressing the soluble FasL form corresponding to the natural cleavage product could not trigger apoptosis and did not induce a neutrophil response. In contrast to the other FasL transfectants, these cells survived as tumor transplants. However, expression of soluble FasL was not benign, but actually suppressed the inflammatory response and protected other transfectants from the effector mechanisms elicted by membrane-bound FasL.
Asunto(s)
Glicoproteínas de Membrana/inmunología , Animales , Sitios de Unión , Membrana Celular/metabolismo , Supervivencia Celular , Proteína Ligando Fas , Femenino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos CBA , Ratones Endogámicos DBA , Ratones Endogámicos MRL lpr , Neutrófilos/inmunología , Peritoneo/citología , Solubilidad , Células Tumorales CultivadasRESUMEN
Human ribosomal protein L7 has been shown to be involved in the control of translation of distinct mRNAs. In this study we report the existence of an RNA carrying sequences that are complementary (antisense) to the entire coding (sense) region of L7 mRNA. L7 antisense transcripts are not polyadenylated and associate with the large subunit of the ribosome. The antisense sequence is encoded by an intronless gene segment distinct from the active copy of the L7 gene. Upon mitogenic stimulation of lymphocytes and monocytes from mice and humans, complex expression patterns of L7 transcripts are observed.